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Compositions and methods for identifying o-linked glycosylation sites in proteins

a technology of o-linked glycosylation and protein, which is applied in the field of protein posttranslational modification, can solve the problems of hampering the understanding of the role of tn in cancer biology and the development, and the identification of its glycosylation site and the carrier protein in the complex sample is highly challenging, and achieves the effect of optimal removal of contaminants

Pending Publication Date: 2022-09-22
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a new technology called EXoO-Tn that allows for the simultaneous tagging of Tn antigen in proteins and mapping of its glycosylation sites using a one-pot reaction. This technology involves the use of two specific enzymes, glycosyltransferase C1GalT1 and OpeRATOR, which work synergistically to add a tag to Tn antigen and release it from solid-phase. The tagged Tn antigen is then cleaved at the N-terminus using an endopeptidase that removes the tag at serine or threonine residues. The resulting peptides are then mapped using liquid chromatography-mass spectrometry. The invention also provides a kit for identifying O-linked glycosylation sites of Tn antigen in proteins, which includes C1GalT1, UDP-Gal, and an endopeptidase. The use of this technology allows for more efficient and accurate identification of O-linked glycosylation sites of Tn antigen in proteins.

Problems solved by technology

Although Tn is structurally simple, identification of its glycosylation sites and the carrier proteins in the complex samples is highly challenging due to the lack of suitable technology.
Limited information regarding Tn-glycosylation sites and carrier proteins hamper the understanding of the role of Tn in cancer biology and the development of new strategies targeting cancers.

Method used

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  • Compositions and methods for identifying o-linked glycosylation sites in proteins
  • Compositions and methods for identifying o-linked glycosylation sites in proteins
  • Compositions and methods for identifying o-linked glycosylation sites in proteins

Examples

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Effect test

example 1

ag-n-Map the Tn Antigen in the Human Genome

[0031]Tn antigen (Tn), a single N-acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser / Thr residues, is found on most solid tumors yet rarely detected in adult tissues, featuring it one of the most distinctive signatures of cancers. Although it is prevalent in cancers, Tn-glycosylation sites are not entirely clear owing to the lack of suitable technology. Knowing the Tn-glycosylation sites will spur the development of new vaccines, diagnostics, and therapeutics of cancers. Here, the present inventors report a novel technology named EXoO-Tn for large-scale mapping of Tn-glycosylation sites. EXoO-Tn utilizes glycosyltransferase C1GalT1 and, in particular embodiments, isotopically-labeled UDP-Gal(13C6) to tag and convert Tn to Gal(13C6)-Tn, which has a unique mass being distinguishable to other glycans. THIS exquisite Gal(13C6)-Tn structure is recognized by a human-gut-bacterial enzyme, called OpeRATOR, that specifically cleaves...

example 2

rotocol. For Cell / Tissue Lysis

[0074]Materials

[0075]Urea (solid) (Sigma U0631-1KG)

[0076]5M NaCl (Santa Cruz Biotechnology, sc-295833)

[0077]1M Tris HCl pH 8.0 (Ambion AM9855G)

[0078]Sequencing grade modified Trypsin (Promega; (V51 IX) Waters tC18 SepPak, 100 mg for desalting of 1-3 mg peptides, 1-3% binding capacity

[0079](Waters; WAT054925)

[0080]C1GalT1 / C1GalT1C1 (R&D Systems)

[0081]UDP-Gal(13C6) (Omicron Biochemicals, Inc.)

[0082]OpeRATOR also called OgpA (Genovis)

[0083]SialEXO (Genovis)

[0084]Trizma hydrochloride solution; pH 7.4, 1M

[0085]DTT (Thermo Fisher Pierce; cat#20291)

[0086]IAA (Sigma; cat# A3221-1OVL or Sigma; cat# Il149-5G)

[0087]Reagent Setup

[0088]8M urea buffer. Fill a 15 ml tube with urea powder to 4.8 g. Add 2 ml 1M TrisHCl pH 8. Fill H2O to the tube to 10 ml mark. Warm the tube in hand to properly dissolve the urea in the buffer. Make fresh before use.

[0089]1M DTT (WM 154.25. 200×) (Thermo Fisher Pierce: cat#20291). Weigh 7.7125 mg. Add 50ul H2O, to make 50ul of solution, m...

example 3

ation of Tn-Glycosylated Markers in Cancer

[0152]EXoO-Tn was performed on sera from individuals with pancreatic cancer. The method identified several Tn-glycosylated proteins including, but not limited to, Tn-glycosylated Kininogen-1 (KNG1), Clusterin (CLU) and Complement Factor H-Related 5 (CFHR5). Accordingly, Tn-glycosylated KNG1, CLU and CFHR5 can be used in methods for diagnosing and / or prognosing pancreatic cancer.

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Abstract

The present invention relates to the field of protein post-translational modification. More specifically, the present invention provides compositions and methods useful for identifying O-linked glycosylation sites in proteins. In one embodiment, the present invention provides a method for identifying O-linked glycosylation sites of Tn antigen in proteins comprising the steps of (a) digesting proteins present in a sample into peptides; (b) enriching for Tn-glycopeptides; (c) conjugating Tn-glycopeptides to solid phase; (d) labeling Tn using the glycosyltransferse enzyme C1GalT1 and a labeled uridine diphosphate galactose (UDP-Gal) substrate to produce labeled Tn-glycopeptides; (e) releasing the labeled Tn-glycopeptides from the solid-phase using an endopeptidase that cleaves peptides at the N-terminus of O-linked glycans at serine or threonine residues; and (f) mapping O-linked glycosylation sites of Tn antigen using liquid chromatography-mass spectrometry.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 62 / 891,497, filed Aug. 26, 2019, which is incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENTAL INTEREST[0002]This invention was made with government support under grant nos. CA210985, Al122382, and CA152813, awarded by the National Institutes of Health. The government 10 has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY[0003]This application contains a sequence listing. It has been submitted electronically via EFS-Web as an ASCII text file entitled “P15799-02_ST25.txt.” The sequence listing is 1,271 bytes in size, and was created on Aug. 21, 2020. It is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION[0004]The present invention relates to the field of protein post-translational modification. More specifically, the present invention provides compositions and methods useful fo...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12N9/10C12N9/76C12P19/18C12P21/00C12P21/06
CPCG01N33/6848C12N9/1051C12N9/6427C12P19/18C12P21/005C12P21/06C12Y204/01C12Y304/21004G01N2440/38
Inventor ZHANG, HUIYANG, WEIMING
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE